Cells, the common building blocks of life, from the single cell to the multi-cellular organism remain functionally intact by virtue of their surrounding envelope. For all cells this is the cell membrane. For specialized cells, i.e. plant cells, this includes a cell wall. To operate in the larger environment cells communicate across the cell membrane, bringing in nutrients, dissolved gases, stimulating biochemicals, signaling molecules, hormones and other ionic or molecular entities involved in function, which may be normal or modified based on these stimuli. Similarly, cells exchange and export numerous compounds which are used for communication, either in the local environment (autocrine and paracrine functions) as well as in the larger environment, i.e. endocrine function or beyond. So the essential element here is the intactness, normality as well as selective function of the cell membrane.
In addition to chemical and biochemical stimuli cells respond to physical stimuli. Application of pressure, electrical fields, magnetic fields, light and other electromagnetic radiation, ultrasound, audible sound and beyond, frictional forces, heat, cold or shear forces may all modulate cell function.
The mechanism by which physical forces, in particular shear forces, tractional forces, frictional forces, and pressure, are sensed by the cell involves a complex set of cellular components via a process referred to as mechano-transduction. Briefly, a series of cell surface receptors, most notably integrins, hetero-dimeric protein receptors made of two subunits, alpha and beta, come together to form a functional receptor. These receptors typically interact with surrounding extracellular matrix proteins and physical substrates.
Integrins represent a complex family with over 18 alpha subunits and 8 beta subunits, which come together to form varying combinations, with a range of specificity to defined matrix proteins. For example, alphal betal and alpha 2 beta 1 typically bind to collagen. Alpha 5 beta-1 more typically binds to fibronectin, alpha V beta 3 has been referred to as the Vitronectin receptor. While elements of specificity exist for specific combinations of integrin subunit pairs, a variable degree of promiscuity exists in integrin-ligand interaction, to provide for redundancy and adhesion. Beyond the adhesive function of integrin receptors, these moieties sit in the membrane like antennae and respond to shear, tugging, and physical manipulation to “transmit” the physical stimuli through the membrane to sub-membrane assemblies.
These sub-membrane assemblies contain protein complexes, which include FAK kinase and other kinases, talin, paxillin and vinculin, all of which function to phosphorylate a cascade of proteins, which then signal, trigger and modulate intra-cellular processes. Such processes may include modulation of protein function, i.e., post-translational modification, induction of protein synthesis or alteration of gene expression. On the physical side integrin-ligand signaling may stimulate intracellular micro filaments, which run throughout the cell like “guywires,” altering cell shape, stimulating cell membrane alteration, sending out cell projections or budding of vesicles. Additionally, microtubules may be stimulated, which act both as physical structural devices as well as transport systems to similarly evoke intra-cellular processes.
A link exists between integrins and the cell membrane. Integrins reside within cell membranes; thus, their positioning, stabilization and grounding within the membrane is essential for normal function in order to transduce mechanical and other physical stimuli.
By analogy, integrins may be viewed as basic pillars or posts within a cell membrane “foundation.” Pillars and posts in the building must be structurally intact to provide support and communication between the building and the underground, so as to provide structural rigidity and stability. If the foundation, i.e. the membrane, in which they reside is shaky, loose or fluid the functional effect, stability in the case of this analogy, is diminished or completely lost. This also applies to integrins and cell membranes. If the cell membrane function is altered due to physical forces, chemical activity or other exogenous stimuli, transduction of information from both the “outside-in,” as well as the “inside-out” may be altered.
Beyond integrin-mediated mechano-transduction, other means of transmitting exogenous physical stimuli into the cell, to alter function, exist. This may occur via perturbation of numerous cell surface receptors as well. These receptors include cadherins, immunoglobulin superfamily, gangliosides, selectins, syndecans and the like.
Further, stimulation may occur via nonspecific membrane damage creating pores, rifts, rents, or other physical communication means allowing contact and mixing of the extracellular and intracellular environments.
A need exists therefore to protect cells from harmful external physical stimuli, to stabilize membrane function so as to limit membrane damage as well as maintain normal integrin function and avoid inappropriate or non-specific mechano-transductive signaling when cells are subjected to exogenous physical stimuli, most notably shear forces, and to otherwise insulate cells to control cell function.
A major disease affecting man today is congestive heart failure. Heart failure is the final common pathway of all forms of heart disease. Whether one is afflicted with atherosclerotic coronary artery disease leading to reduced blood flow in coronary vessels; or one has a myopathy in which the cardiac muscle pumps poorly; or suffers from an arrhythmia where cardiac contraction is cacophonous; or has a narrowed (stenotic) or insufficient valve, at the end of the day heart pump function is compromised. This is known as heart failure. While we have made major advances in the treatment of heart failure through the use of pharmacologic agents such as ACE inhibitors, beta blockers, digoxin and diuretics, as heart function declines one eventually needs augmentation or replacement of pump function. This need has led in recent years to the advance of a new field known as mechanical circulatory support or MCS. In MCS a series of pump devices including ventricular assist devices (VADs) as well as the total artificial heart have been developed. While these devices are effective in either augmenting or supplanting cardiac function, at the same time they subject blood cells to significant exogenous physical forces. The most extreme example of this phenomenon occurs with ventricular assist devices.
As blood traverses and is propelled through a VAD, significant shear forces are imparted to the cells. In the beginning of the MCS era, devices were largely pulsatile, subjecting cells to shear and turbulence at a level above physiologic shear (i.e. >0-50 dynes/cm2) though typically in ranges only slightly above physiologic levels. A new class of VAD was developed, that of a continuous flow rotary or centrifugal blood pump. These devices are akin to a small “jet engine” being placed within the bloodstream. They spin at ranges of 7000 to 12,000 rpm or beyond and impart shear in the 100's if not 1000's of dynes/cm2. As such platelets, red blood cells and white cells subjected to these extreme forces undergo physical stimulation via both mechano-transductive mechanisms as well as direct force interaction leading to both biochemical and physical stimulatory events. In the extreme, these forces may lead to membrane fragmentation, resulting in direct communication between the extracellular and intracellular environment, or frank disruption of these cells leading to spillage of intracellular contents and exposure of cell membranes in the bloodstream. In the blood environment this can have dire if not fatal consequences.
This type of stimulation of platelets, (as well as RBCs and WBCs) instantly and dramatically drives thrombosis. If the level of damage and the number of cells stimulated is significant enough both local and distant thrombus formation may occur. This can lead to reduction of VAD pump function and potential death of the patient due to reduced cardiac output. More commonly thrombus formation may propagate, break off, embolize, or be showered distally via the pump, leading to embolic consequences including stroke and transient ischemic attack (TIA), and coronary, renal, peripheral or other forms of infarction.
Presently, conventional anti-platelet agents such as aspirin, dipyridamole, pentoxyfylline, theinopyridines, CPTP inhibitors and anti-coagulants, such as heparin, warfarin, rivaroxiban, and direct thrombin inhibitors are used to limit platelet activation and associated biochemical coagulation—involving the intrinsic, extrinsic and common pathways. While these drugs are known to be effective for conventional biochemical stimulation of platelets and coagulation under conditions of low shear, stagnation and pooling, in recent studies it has been demonstrated that these agents have limited or no effect under the “hyper shear” conditions experienced with MCS systems.
As such a need exists to stabilize these cells and protect, insulate and otherwise limit the impact of their high propulsion flow and subsequent sheer on membrane intactness and transmembrane signaling.
Therefore it is an object of the invention to provide compositions, kits, devices, and methods for protecting or insulating cells or to otherwise limit the impact of exogenous physical stimuli on the cells.
It is a further object of the invention to provide improved methods for using devices that apply exogenous physical stimuli on the cells to limit the impact of exogenous physical stimuli on the cells.